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CRISPR/Cas base editors offer precise conversion of single nucleotides without inducing double-strand breaks. This technology finds extensive applications in gene therapy, gene function analysis, and other domains. However, a crucial challenge lies in selecting the appropriate guide RNAs (gRNAs) for base editing. Although various gRNAs design tools exist, creating a simplified base-editing library with diverse protospacer adjacent motifs (PAM) sequences for gRNAs screening remains a challenge. We present a user-friendly web tool, BES-Designer ( https://bes-designer.aielab.net ), for gRNAs design based on base editors, aimed at streamlining the creation of a base-editing library. BES-Designer incorporates our proposed rules for target sequence simplification, helping researchers narrow down the scope of biological experiments in the lab. It allows users to design target sequences with various PAMs and editing types simultaneously, and prioritize them in the simplified base-editing library. This tool has been experimentally proven to achieve a 30% simplification efficiency on the base-editing-library.
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http://dx.doi.org/10.1007/s12539-024-00663-6 | DOI Listing |
Microbiol Spectr
September 2025
Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, China.
Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in and , it remains highly inefficient in many nontuberculous mycobacteria (NTM), including . Here, we discovered that NTM such as exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage.
View Article and Find Full Text PDFExp Mol Med
September 2025
Department of Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
RNA modifications add a crucial layer to gene expression regulation, though the roles of many RNA modification-related genes in cancer remain largely unexplored. Here we profile 76 RNA modification-associated genes across nine distinct types of modification (N-methyladenosine, 5-methylcytosine, N,2'-O-dimethyladenosine, 2'-O-dimethyladenosine, N-methylguanosine, pseudouridine, uridylation, 2'-O-methylation, N-acetylcytidine and adenosine-to-inosine editing) in four cancer types-breast, colon, liver and lung-through a comprehensive analysis of The Cancer Genome Atlas data. Our analysis identified three candidate genes with increased expression in cancer tissues, with elevated levels associated with poor survival across multiple cancer types: the 5-methylcytosine methyltransferases NSUN2 and DNMT3B and CBP20, an N-methylguanosine binding protein.
View Article and Find Full Text PDFBiomolecules
August 2025
Department of Life Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy.
The tumor suppressor DAB2IP, a RasGAP and cytoplasmic adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radioresistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms.
View Article and Find Full Text PDFBiomolecules
August 2025
Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, Northeast Agricultural University, Harbin 150030, China.
The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability.
View Article and Find Full Text PDFNat Commun
August 2025
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features.
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